This invention generally relates to the nucleic acid sequences (and corresponding translated products) of novel mutant forms of the Drosophila DIAP1 gene and methods of identifying and testing agonists and antagonists of DIAP1 that enhance or reduce the apoptotic process.
Essentially all animal cells have the ability to activate an intrinsic cell suicide program, called programmed cell death (Steller, H. xe2x80x9cMechanisms and Genes of Cellular Suicidexe2x80x9d, Science 267:1445-1446, 1995; White E. xe2x80x9cLife, death and the pursuit of apoptosisxe2x80x9d, Genes Dev. 10:1-15, 1996; Jacobson, M. D., et al. xe2x80x9cProgrammed Cell Death in Animal Developmentxe2x80x9d, Cell 88:347-354, 1997). The execution of this program leads to a morphologically distinct form of cell death termed apoptosis (Kerr et al. xe2x80x9cApoptosis: a basic biological phenomenon with wide ranging implications in tissue kineticsxe2x80x9d, Br. J. Cancer 26:239-257, 1972; Wyllie et al. xe2x80x9cCell Death: the significance of apoptosisxe2x80x9d, Int. Rev. Cytol.xe2x80x9d 68:251-306, 1980). It is now generally accepted that apoptosis is of central importance for the development and homeostasis of metazoan animals. The roles of apoptosis include the sculpting of structures during development, deletion of unneeded cells and tissues, regulation of growth and cell number, and the elimination of abnormal and potentially dangerous cells. In this way, apoptosis provides a stringent and highly effective xe2x80x9cquality control mechanismxe2x80x9d that limits the accumulation of harmful cells, such as self-reactive lymphocytes, virus-infected cells and tumor cells (Reed xe2x80x9cRegulation of apoptosis by bcl-2 family proteins and its role in cancer and chemoresistancexe2x80x9d, Curr. Opin. Oncol 7:541-546, 1995; Thompson xe2x80x9cApoptosis in the Pathogenesis and Treatment of Diseasexe2x80x9d, Science 267:1456-1462, 1995; Naik et al. xe2x80x9cThe rise and fall of apoptosis during multistage tumorigenesis: down-modulation contributes to tumor progression from angiogenic progenitorsxe2x80x9d Genes Dev. 10:2105-2116, 1996; Morin et al. xe2x80x9cApoptosis and APC in colorectal tumorigenesisxe2x80x9d, Proc. Natl. Acad. Sci. USA 93:7950-7954, 1996; White xe2x80x9cLife, death and the pursuit of apoptosisxe2x80x9d Genes Dev. 10:1-15. 1996). On the other hand, inappropriate apoptosis is associated with a wide variety of diseases, including AIDS, neurodegenerative disorders, and ischemic stroke (Martinou et al. xe2x80x9cOver-expression of Bcl-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemiaxe2x80x9d Neuron 13:1017-1030, 1994; Thompson xe2x80x9cApoptosis in the Pathogenesis and Treatment of Diseasexe2x80x9d, Science 267:1456-1462, 1995; Pettmann and Henderson xe2x80x9cNeuronal Cell Deathxe2x80x9d Neuron 20:808-810, 1998).
Because it is now clear that apoptosis is the result of an active, gene-directed process, it should be possible to manipulate this form of death by developing drugs that interact with cell death proteins. Prior attempts at drug screening have been hampered by the lack of reagents that allow for the identification of compounds that interact with known regulatory constituents of the cell death mechanism. There is a pressing need for new reagents that help identify cell death agonistic or antagonistic compounds that act with specificity at known cell death modulating proteins. Knowing specifically where the compounds interact in the cell death pathway will allow for the modification of those compounds found to be agonistic or antagonistic thereby allow for the development of improved versions of the compound.
The present invention generally relates to compositions and methods of identifying and testing DIAP1 pathway agonists and antagonists. In addition, the invention relates to methods to identify other members of the DIAP1 signal pathway, methods to identify homologs of DIAP1 which are native to other tissue or cell types, methods to identify tissues that may harbor tumors expressing similar or homologous genes with simular mutations and methods to generate reagents derived from the invention.
The present invention contemplates employing novel mutant forms of the wild-type Drosophila DIAP1 gene (SEQ ID NO:1) in these screening methods. In one embodiment, the present invention contemplates generating chemically induced mutants that modulate the partial eye abolation phenotype of the GMRreaper and/or GMRHid of trangenic Drosophila melanogaster. In this way it is possible to screen for gof and lof mutations. In one embodiment, the present invention contemplates a composition comprising isolated and purified DNA having an oligonucleotide sequence selected from the group consisting of: DIAP16-3S cDNA having the nucleotide sequence of SEQ ID NO: 2; DIAP145-2S cDNA having the nucleotide sequence of SEQ ID NO: 3; DIAP123-4S cDNA having the nucleotide sequence of SEQ ID NO: 4; DIAP111-3E cDNA having the nucleotide sequence of SEQ ID NO: 5; DIAP122-8S cDNA having the nucleotide sequence of SEQ ID NO: 6; DIAP121-4S cDNA having the nucleotide sequence of SEQ ID NO: 7; DIAP133-1S cDNA having the nucleotide sequence of SEQ ID NO: 8; DIAP121-2S cDNA having the nucleotide sequence of SEQ ID NO: 9; DIAP141-8S cDNA having the nucleotide sequence of SEQ ID NO: 10. Such DNA may readily be inserted into expression constructs and the present invention contemplates such constructs as well as their use. The present invention also contemplates RNA transcribed from the above-indicated cDNAs as well as protein (typically purified protein) translated from this RNA. Moreover, the present invention contemplates antibodies produced from immunizing with this translated protein.
The present invention also contemplates transgenic animals comprising the above-indicated DNA (i.e. the xe2x80x9ctransgenexe2x80x9d) or portions thereof. In a particular embodiment, the transgenic animal of the present invention may be generated with the transgene contained in an inducible, tissue specific promotor.
The present invention also contemplates using the above-named compositions in screening assays. The present invention is not limited by the particular method of screening. In one embodiment insect cells are used such as, but not limited to, Drosophila SL2 cells. In another embodiment mammalian cells may be used. The present invention is not limited to the nature of the transfection construct. The transfection constructs utilized will be the optimal constructs available for the cell line chosen at the time of setting up the assay. In one embodiment, the present invention contemplates screening suspected compounds in a system utilizing transfected cell lines. In one embodiment, the cells may be transfected transiently. In another embodiment, the cells may be stably transfected. In yet another embodiment translation products of the invention may be used in a cell-free assay system. In yet another embodiment, antibodies generated to the translation products of the invention may be used in immunoprecipitation assays.
The present invention may also be used to screen for tumors which manifest mutations in genes similar to, or homologous with, the cDNA encoding the invention. In, one embodiment cDNA encoding the invention may be used in microchip assays. The present invention contemplates a method of screening, comprising: a) providing in any order: i) a first solid support (e.g. microchip) comprising cDNA encoding at least a portion of the oligonucleotide sequence of SEQ ID NOS: 2, 3, 4, 5, 6, 7, 8, 9 or 10, ii) a second solid support (e.g. a second microchip) comprising at least a portion of the wild type Drosophila DAIP1 gene oligonucleotide sequence (SEQ ID NO:1), and iii) sample DNA from at least one tissue sample suspected of having mutations in genes similar to (or homologous with) SEQ ID NOS: 2, 3, 4, 5, 6, 7, 8, 9 or 10; b) contacting said first and second microassay microchips with said sample DNA under conditions such that hybridization can take place.
The present invention may also be used to identify new constituents of the DIAP1 signaling pathway. In one embodiment, antibodies generated to translation products of the invention may be used in immunoprecipitation experiments to isolate novel DIAP1 pathway constituents or natural mutations thereof. In another embodiment, the invention may be used to generate fusion proteins that could also be used to isolate novel DIAP1 pathway constituents or natural mutations thereof. In yet another embodiment screens may be conducted using the yeast two-hybrid system.
The present invention may also be used to identify new homologs of DIAP1 or natural mutations thereof. The present invention contemplates screening for homologs using standard molecular procedures. In one embodiment screens are conducted using Northern and Southern blotting.
The present invention contemplates a method of screening a compound, said method comprising: a) providing in any order: i) a first group of cells comprising a recombinant expression vector, wherein said vector comprises at least a portion of the oligonucleotide sequence of SEQ ID NOS: 2, 3, 4, 5, 6, 7, 8, 9 or 10, ii) a second group of cells comprising a recombinant expression vector, wherein said vector comprises at least a portion of the wild-type Drosophila DIAP1 gene oligonucleotide sequence (SEQ ID NO:1), and iii) at least one compound suspected of having the ability to modulate DIAP1 pathway activity; b) contacting said first and second groups of cells with said compound; and c) detecting programmed cell death modulation effects of said compound.
The present invention also contemplates a method of screening for homologs, said method comprising: a) providing in any order: i) first nucleic acid comprising at least a portion of the sequence of SEQ ID NOS: 2, 3, 4, 5, 6, 7, 8, 9 or 10, ii) second nucleic acid comprising at least a portion of the sequence of SEQ ID NO:1; and iii) DNA libraries from cells or tissues suspected to comprise said homolog; and b) hybridizing said first or second nucleic acid with said DNA of said library under conditions such that said DNA suspected of coding for said homolog is detected.
The present invention also contemplates a method of screening for interactive peptides, said method comprising: a) providing in any order: i) a peptide comprising at least a portion of the peptide sequence of SEQ ID NOS: 12, 13, 14, 15, 16, 17, 18, 19 or 20 (including but not limited to portions that are part of fusion proteins, i.e. proteins that contain another portion, such as a portion useful for protein purification) and b) an extract from source (e.g. cells or tissues) suspected of having said interactive peptides; and c) mixing said peptide with said extract under conditions such that said interactive peptide is detected.
The present invention contemplates another approach for screening for interactive peptides, said method comprising: a) providing in any order: i) antibodies reactive with (and usually specific for) at least a portion of a peptide having the sequence of SEQ ID NOS: 12, 13, 14, 15, 16, 17, 18, 19 or 20, and ii) an extract from a source (e.g. cells or tissues) suspected of having said interactive peptide(s); and b) mixing said antibody with said extract under conditions such that said interactive peptide is detected.